The viscosity of a fluid may be measured using a viscometer. Viscometers measure the properties of a fluid under conditions where the fluid remains stationary and an object, such as a vibratory element moves through it. The measurement of the viscosity can be determined based on the drag caused by the relative motion of the fluid and surface.
One specific type of viscometer is a vibration viscometer. Vibration viscometers typically function by allowing the measurement of the dampening of an oscillating electrochemical resonator which is immersed in the fluid of which the viscosity is to be determined. The greater the viscosity of the fluid, the larger the dampening effect imposed on the resonator.
A change in the density or viscosity of a fluid can accompany or be the result of the occurrence of a chemical reaction. A change in the density or viscosity of a test sample can, in particular, occur during the performance of assays commonly used in areas such as haematology where blood clotting, or coagulation reactions are measured, in immunology where immunoassays are performed, and further for more general screening, such as high throughput screening, for example to detect the presence of a contaminant or analyte in a sample.
One example of such an assay which uses a change in density or viscosity of a test sample in order to provide result is assay based test to determine the presence of bacterial endotoxin in a test sample.
Bacterial endotoxin, such as lipopolysaccaharide (LPS), is a fever producing product of gram negative bacteria. Endotoxins typically have a hydrophobic core which is bound to repeating oligosaccharide side chains. When present in the bloodstream, even in low levels, endotoxins can cause fever, shock, hypotension, raised erythrocyte count, and disseminated intravascular coagulation. If the endotoxin is present in a sufficient high level in the bloodstream, it can cause death.
The US Food and Drug Administration department (FDA) requires drugs and pharmaceutical compounds which are to be administered to a subject either by injection, or intravenously, to undergo an endotoxin test prior to their administration. Furthermore, prosthetic devices, such as heart valves or hip replacements also require such an endotoxin test. There is therefore an on-going need to provide accurate testing to determine the presence of endotoxin within a test sample.
The traditional test used to identify the presence of pyrogens, such as endotoxin, was the rabbit pyrogen test. This test, which dates back to 1942, suffered the disadvantages of being both slow and expensive to perform. Subsequent to this, Levin and Bang discovered that blood cells (amebocytes) which were derived from the horseshoe crab (Limulus polyphemus), contained a clotting agent that attaches to the endotoxins produced by gram-negative bacteria. This clotting agent was identified as Limulus amoebocyte lysates, which is commonly abbreviated to “LAL”.
LAL was used to develop a number of gellation based clotting tests which detect and quantify the presence of endotoxin within a test sample. A number of such LAL-based tests are currently used commercially to determine the presence and quantity of endotoxin within a sample.
For example, the gel-clot test is a sample test that uses a lysate preparation of Limulus polyphemus blood to give a positive/negative answer by means of the presence or absence of gel-clot formation within a test tube or vial. The presence of endotoxin results in clotting, and hence, this change of state can be visually determined by inverting the vessel in which the reaction has occurred.
Preparations of LAL lysate are commercially available in different sensitivities, to give a qualitative. In addition, testing kits that contain LAL reagents may be provided in ‘ready to use’ tube vials, which allow for diluted samples to be added to the vials so that the assay can be performed within them.
Gel-clot kits are frequently used for field testing. They may also be used in laboratories where small sample throughput is required. The maximum sensitivity of the gel-clot test is generally a level of around 0.03 EU/ml of endotoxin in a sample. However, there are disadvantages associated with such tests, in particular the cost as a large quantity of LAL reagent must be used for each sample. Furthermore, the results from the gel-clot LAL test are subject to human interpretation, as the results are visually determined. An assay based on the subjective determination of a gel-clot is particularly difficult to automate, hence determining the result of the gel-clot test in this way may introduce a greater degree of error into determining the results of the test, and may further lead to the increased risk of reporting a false negative result.
A further technique used to identify endotoxin in a sample is by means of optical analysis: chromogenic or turbidometric methods. These methods can be fully automated, and further have the advantage of providing quantitative results. These tests can also use a “kinetic” approach, measuring a response as a function of time can be obtained, rather than waiting for the entire reaction to complete before determining the result.
The turbidometric method is a sensitive LAL-based method used for determining endotoxin levels in a sample, with a maximum sensitivity of 0.001 EU/ml. However, for this level of sensitivity the methodology requires the use of costly, large and sensitive laboratory equipment, and therefore analysis of this type is restricted. Equipment which allows for “kinetic” LAL testing to be performed on a smaller scale, albeit with lower sensitivity is commercially available; however such systems (for example ENDOSAFE™, Charles River Laboratories) use microfluidics and optical technology to analyse the sample within a tiny sampling area. Such approaches have limited sensitivity and poor precision due to the short pathlength of the interrogating light through the sample. The resulting method will be suffer increases in analysis time and decreases in precision with a desirable reduction in sample volume that enable reductions in reagent and sample usage
A quartz crystal microbalance (QCM) may be used for detecting the end-point of a gellation or agglutination reaction. For example a LAL assay can be performed by immersing the crystal within a test sample undergoing a LAL reaction. A key failing of resonating crystals is caused due to the high frequency of resonance of a pure piezoelectric resonator (for example in European Patent No 0,304,283 B1 these values are in the order of 6-12 MHz). The depth of the liquid probed by the vibration will therefore be very small, and so it is to be expected that the signal will also be very small. However, WO 2005/114138 teaches that applying a texture to the surface of a planar resonating device operating at a megahertz frequency improves the viscosity and density measuring capability. The textured surface creates an entrapped layer of liquid close to the sensor surface. In the case of monitoring of biological reactions, this not ideal because the sensor now responds according to the kinetics of the diffusion of the reagents and reaction products in the entrapped layer, rather than responding to the reaction in the bulk. In order to address these shortcomings in the devices known in the prior art, the inventors have identified that ideally a lower frequency resonator, for example of 400 kHz or less, would be optimal for the measurement of biological reactions.
U.S. Pat. No. 6,401,519 teaches how mechanical oscillators can be used to analyse the properties of fluids. Different viscosities and density values present different characteristic frequency/amplitude responses. A twin beam cantilevered tuning fork as shown in U.S. Pat. No. 6,401,519 suffers from energy losses due to moments acting at the mountings of the beams. The clamping/mounting of electrodes or coatings at the mountings of the beams will create wide variations in sensor performance. The high-loss twin beam cantilever tuning fork has little energy to resonate in liquid and is almost completely damped. Furthermore, the twin cantilevered arrangement disclosed cannot be reliably waterproofed as coating the mountings cause this to be damp the signals further.
Following extensive experimentation, the present inventors have surprisingly provided an improved apparatus and method for use in performing real time monitoring of the progress of an assay or chemical reaction, wherein the assays or chemical reaction results in a change or density or viscosity of a test sample, typically by way of the production of a gel, precipitate, coagulate, precipitate or the like. In particular, the inventors have provided an apparatus and method for the detection of endotoxin in a test sample using amoebocyte lysates, wherein the progress of the gellation of the test sample can be monitored in real time. The methods of the invention are advantageous in as far as they provide a quantitative assay result, and as such, when employed in relation to assays, such as the determination of bacterial endotoxin contamination in a sample, they provide the further associated advantages of an enhancement in sensitivity, specificity and throughput, when compared to LAL-based endotoxin tests currently known in the art.